(a) Field of the Invention
The present invention relates to a FabK (enoyl-acyl carrier protein reductase) protein derived from a Thermotoga maritima strain. In the present invention, the active site and the three-dimensional crystal structure of the protein are determined, a novel inhibitor against the FabK protein is screened and/or designed using the three-dimensional crystal structure thereof, and thereby developing a novel active compound, namely active-controlling material with excellent antibiotic activities against strains having a resistance to previous antibiotics.
(b) Description of the Related Art
Triclosan, which is known as an antibacterial agent having comprehensive antibacterial activities, has been used to give antibiosis and antibacterial activities to various products for the past 30 years. For example, the triclosan is included in various products such as soaps, shampoos, washing agents, dentifrices, cosmetics, toys, rugs etc. Therefore, the category of usage of triclosan is the same as for ultraviolet or bleach.
Triclosan inhibits the activity of enzymes called enoyl-[acyl-carrier-protein] reductase (E.C. 1.3.1.9), which is also known as NADH-dependent trans-2-enyol-ACP reductase (ENR), or FabI. FabI exists in almost all bacteria and fungi, and it is an essential enzyme for living microorganisms since it is one of enzymes involved in biosynthesis of fatty acid and fatty acid is essential for forming the cell membrane. Therefore, triclosan is used in a wide range of biocides.
In the case of mycobacteria of an aerobic bacteria, the enzyme that carries the action of the enoyl-ACP reductase or FabI is called InhA. The two enzymes, InhA and FabI, shares rather low sequence identities, but the key residues at the active site of the two enzymes are the same. Thus, InhA shares the same enzymatic mechanism as FabI. Isoniazid, the existing antitubercular agent, inhibits InhA in the same manner as triclosan inhibiting FabI.
Fatty acids are central building blocks of life, since they are constituents of cell membranes, energy storage compounds, and messenger substances, and they act as posttranslational protein modifiers and modulate gene expression. Therefore, the de Novo synthesis of fatty acid is essential for all living organisms. It involves a conserved set of chemical reactions for the cyclic stepwise elongation of activated precursors by two carbon units. The growing fatty acid is attached to a carrier protein, acyl carrier protein (ACP), throughout its synthesis and is released once it reaches certain length.
Although all organisms use variations of this common synthetic scheme, it appears that there are three distinct architectures for fatty acids synthesis. In bacteria, all reactions are carried out by individual, mono-functional proteins in a dissociated manner. On the contrary, the eukaryotic type I FAS consists of large multifunctional polypeptides. Fungal FAS is a 2.6MD α6β6 dodecamer, while the FAS of vertebrates and mammals are a α2 homodimer of a single 270-kDa polypeptide. FabI manages the process that reduces the Enoyl-ACP corresponding to the final step among the steps, and then the reduced Enoyl-ACP is converted into Acyl-ACP.
As described in above, FabI performs an essential role for the biosynthesis of fatty acids by catalyzing the elongation of the lipid in the final step of the biosynthesis of lipids. FabI belongs to the short-chain alcohol dehydrogenase/reductase (SDR) superfamily, and it forms a tetramer like other family members. In FabI isolated from E. coli, the FabI uses the coenzyme called NADH [Egan, A. F. and Russel, R. R. B., Genet. Res., 21, 3603-3611 (1973)]. Recently, according to the FabI-triclosan complex structure, the triclosan is located at the upper part of the NAD+ of NADH, to occupy the active site of the FabI enzyme, even though triclosan is non-covalent, because it has quite strong binding activity with the FabI enzyme, and thereby the function of the enzyme is inhibited [Heath, R. J., et al., J. Biol. Chem., 274, 11110-11114 (1999)]. In the case of a staphylococcus aureus, it is clarified that FabI uses NADPH as a coenzyme but not NADH, and the triclosan inhibits the activity of the FabI enzyme through an interaction similar to the above. Therefore, FabI inhibitors, which are being developed in universities and various research institutes as well as by=pharmaceutical companies, are compounds having a mechanism similar to triclosan.
But recently, it was reported that a part of major pathogens, such as Streptococcus pneumoniae, have resistance against triclosan [Heath, R. J. and Rock, C. O., Nature, 406, 145-146 (2000)]. In these resistant strains, although the FabI enzyme did not exist in the strains, because a FabK gene exists in the gene cluster of the strains, it was clarified that the FabK protein is expressed and produced by the FabK gene, and it performs a role identical to the FabI protein. It was reported that strains including Enterococcus faecalis and Thermotoga maritima use also FabK instead of FabI in the pathway of biosynthesis of fatty acids. The gene sequence coding the FabK protein is different from the gene sequence coding the previous FabI protein, and does not have any similarity with the gene sequence coding other proteins of the SDR superfamily. In FabK existing in strains, such as Enterococcus faecalis and Thermotoga maritima, the amino acid sequence of the FabK has the sequence identity of 68% and 48% when compared with the FabK amino acid sequence of Streptococcus pneumoniae, respectively [Marrakchi, H., et al., Biochem. J., 370, 1055-1062 (2003)]. Therefore, FabK is a flavoprotein, it needs NADH as a coenzyme for its activity, and it is a protein that is not deactivated by triclosan.
Because the current antitubercular agents mostly relate to FabI/InhA, strains having resistance to the antitubercular agents through not using FabI/InhA are often being discovered. These strains use FabK instead of FabI. For example, it has been clarified that S. pneumoniae of a major strain of respiratory disease or E. facalis having a resistance against vancomycin uses also FabK instead of FabI. Namely, in order to have antibiotic activities against the resistant strains, a novel compound inhibiting the activity of FabK, not FabI, is necessary. According to reports up to now, the mechanism of operating FabK differs from the mechanism of operating FabI. Therefore, research for the structure and the function of FabK is acutely required.